Current genetic models for studying congenital heart diseases: Advantages and disadvantages

Congenital heart disease (CHD) encompasses a diverse range of structural and functional anomalies that affect the heart and the major blood vessels. Epidemiological studies have documented a global increase in CHD prevalence, which can be attributed to advancements in diagnostic technologies. Extensive research has identified a plethora of CHD-related genes, providing insights into the biochemical pathways and molecular mechanisms underlying this pathological state. In this review, we discuss the advantages and challenges of various In vitro and in vivo CHD models, including primates, canines, Xenopus frogs, rabbits, chicks, mice, Drosophila, zebrafish, and induced pluripotent stem cells (iPSCs). Primates are closely related to humans but are rare and expensive. Canine models are costly but structurally comparable to humans. Xenopus frogs are advantageous because of their generation of many embryos, ease of genetic modification, and cardiac similarity. Rabbits mimic human physiology but are challenging to genetically control. Chicks are inexpensive and simple to handle; however, cardiac events can vary among humans. Mice differ physiologically, while being evolutionarily close and well-resourced. Drosophila has genes similar to those of humans but different heart structures. Zebrafish have several advantages, including high gene conservation in humans and physiological cardiac similarities but limitations in cross-reactivity with mammalian antibodies, gene duplication, and limited embryonic stem cells for reverse genetic methods. iPSCs have the potential for gene editing, but face challenges in terms of 2D structure and genomic stability. CRISPR-Cas9 allows for genetic correction but requires high technical skills and resources. These models have provided valuable knowledge regarding cardiac development, disease simulation, and the verification of genetic factors. This review highlights the distinct features of various models with respect to their biological characteristics, vulnerability to developing specific heart diseases, approaches employed to induce particular conditions, and the comparability of these species to humans. Therefore, the selection of appropriate models is based on research objectives, ultimately leading to an enhanced comprehension of disease pathology and therapy.


Background:
Structural or functional abnormalities in the heart or major vessels at birth are characteristic of congenital heart disease (CHD).These anomalies are attributed to genetic variation, environmental influences, or a combination of both elements [1].The most common type of birth defect is congenital heart defect [2].The prevalence of CHD is on the rise, reaching 9.41 per 1000 live births during the previous 15 years, signifying a substantial escalation in the global impact of CHD [3].Various factors influence documented birth prevalence, including the definition of CHD, diagnostic capacity, screening and detection methods, and administrative considerations, such as diagnosis registration.Giang et al. identified ethnicity and genetic, environmental, and socioeconomic factors as potential additional variables influencing birth prevalence [4].A recent study documented geographical disparities in the prevalence of CHD, with the lowest and highest rates in Africa and Asia, respectively [3].Congenital cardiac defects can be classified into various categories, which can be employed to highlight the fundamental anatomical and pathophysiological aspects.These defects can be classified into four main categories: CHD characterized by a shunt between the systemic and pulmonary circulation, CHD associated with left or right heart issues, CHDs involving the aberrant origin of the major arteries, and CHD accompanied by other coexisting disorders [5].CHD continues to be a significant contributor to both mortality and morbidity among individuals across their lifespan, including children and adults [6].Congenital arrhythmias can be potentially lifethreatening and lead to abrupt cardiac death [7].CHD can be hereditary or non-genetic.Despite decades of international efforts to address these factors, the number of nongenetic causes of CHD is still expanding and changing.Dioxins, pesticides, and polychlorinated biphenyls are environmental factors.In addition, CHD can be caused by maternal exposure to alcohol, isotretinoin, thalidomide, and antiseizure medications.Other CHD risk factors include taking antiretroviral medications and obesity associated with diabetes mellitus and hypercholesterolemia [8].Evidence supporting genetic underpinnings of CHD is multifaceted.A higher concordance in monozygotic twins than in dizygotic twins indicates a genetic predisposition, even as twinning itself emerges as a modest risk factor for CHD [9].The recurrence risk among siblings for related and discordant forms of CHD further underscores genetic influences.A minority of rare Mendelian forms of CHD offer crucial insights into conditions, such as atrial septal defects, heterotaxy, mitral valve prolapse, and bicuspid aortic valve [9].Intriguingly, CHD cases within families without a history of CHD significantly contribute to de novo genetic events including chromosomal abnormalities, copy number variants (CNVs), and point mutations.A noteworthy aspect of CHD is its increased prevalence in populations characterized by high consanguinity, implying the involvement of recessive genetic factors.Genetic factors play a significant role in the etiology of CHD considering the potential interplay between genetics and environmental triggers [9].The accurate determination of the genetic factors responsible for heart abnormalities is challenging.This is primarily due to the complex nature of the genetic network that governs heart development [10].As mentioned in the Introduction, the genetics of CHD are heterogeneous [11].
According to epidemiological research, the prevalence of singlegene disorders in individuals with CHD as part of a syndrome ranges from 3% to 5%.Moreover, gross chromosomal aberrations or aneuploidy are detected in approximately 8-10% of individuals with CHD as part of a syndrome, whereas pathogenic CNVs are observed in 3-25% of the same population.Among individuals with isolated CHD, the prevalence of pathogenic CNVs ranged from 3% to 10%.[12].Extensive genetic analysis of CHD using next-generation sequencing (NGS) indicated that approximately 8% and 2% of the cases can be attributed to de novo autosomal dominant and inherited autosomal recessive variations, respectively [13].Although diligent endeavours have been made in this field, the precise genetic pathways underlying CHD remain inadequately understood, and an estimated 55% of individuals affected by CHD do not have a genetic diagnosis [14].Yasuhara and Garg summarized non-syndromic (Table 1) and syndromic (Table 2) CHD-associated genes [15].Researchers have developed several models to understand the genetic factors associated with CHD and identify the genes responsible for its occurrence.In this review, we aimed to highlight the most common in vivo and in vitro models, and how these models were employed to validate the causative genes of CHD in humans (Figure 1).

Xenopus:
Xenopus frogs, notably Xenopus laevis and Xenopus tropicalis, offer versatile and efficient in vivo systems for investigating human diseases.These species are valuable models with unique strengths, which can be tailored to specific research approaches.Although Xenopus species possess distinct attributes, they share key experimental advantages that have made them pivotal in embryology.The ability to breed Xenopus year-round, yielding substantial clutch sizes of up to 2000 eggs per frog per day, coupled with straightforward in vitro fertilization, ensures a continuous supply of developmentally synchronized embryos.These embryos undergo external development, rendering them accessible for microinjection-based genetic manipulation.With its uncomplicated husbandry, Xenopus has emerged as an affordable and practical model for large-scale experiments, including screening and characterizing candidate genes related to human diseases.The frog model has been instrumental in employing genetic knockdown approaches such as morpholino (MO)s and mRNA overexpression of well-known diseaseassociated genes in embryonic development [24].Moreover, the cardiac morphology of Xenopus has a greater resemblance to that of humans than that of fish.For example, Xenopus shares certain characteristics with humans, including the atrial septation.In addition, Xenopus possesses a comparatively compact diploid genome, measuring approximately 1.5 GB in size.This compact genome retains a significant degree of synteny with the human genome, thereby facilitating the identification of orthologous genes.Furthermore, the capacity to generate a substantial number of embryos and the lack of recent genome duplications has enhanced the feasibility of employing MO knockdown technology for screening purposes [24].Xenopus continues to illuminate the complexities of CHD, contributing to advancements in our understanding of its critical conditions.The genes that were characterized and validated using the Xenopus model are summarized in Table 3 [25].
Although Xenopus is widely recognized as a valuable model organism, it has several limitations that impede its utility in genetic studies.Initially, it was noteworthy that X. laevis could be classified as a pseudo-tetraploid because of an extra genome duplication event that occurred approximately 30 million years ago, which distinguished it from other vertebrates.In addition to the increased genome size associated with pseudotetraploidy, the likelihood of successful mutagenesis screening was diminished because of the functional redundancy observed among closely related paralogous genes.One notable drawback of X. laevis is its comparatively long generation time, typically spanning 1-2 years.Consequently, the process of generating stable transgenic lines is hindered at a slow pace [26].

Rabbits:
Rabbits (Oryctolagus cuniculus) exhibit cellular electrophysiology and Ca 2+ transport that resembles those observed in humans to a greater extent than in rats or mice.Alterations in ion channels and calcium transporters are anticipated to directly affect contractile function and the occurrence of arrhythmias, rendering them of considerable importance in the study of heart failure (HF) and arrhythmias.The ventricular action potentials (APs) of mice and rats are characterized by their brevity and the absence of the prominent AP plateau phase observed in humans, rabbits, and larger mammals.Animal transgenesis has led to significant advancements in the replication of human cardiac diseases in rabbits [27].Significant progress has been made in transgenic research with the successful creation of an initial Short QT syndrome (SQT1) transgenic rabbit model [28].This model effectively replicated the phenotypic characteristics of the corresponding human disease across several levels, including ion current, cellular, tissue, whole-heart, and in vivo simulations, specifically in the ventricles and atria.The model overexpresses a disease-specific human mutation (KCNH2/HERG-N588K5) under the control of the rabbit β-myosin-heavy-chain-promoter in the heart without concomitant structural alterations, and thus has no confounding effects on electrical features and arrhythmogenesis [28].Despite this advancement, we should consider that the results may not be transferred across species, and more funds are needed to create transgenic control rabbits with inert genes [29].Other disadvantages include lower efficacy of genetic manipulation, lower reproduction rates, and relatively higher housing/breeding costs [27].

Chicken:
Chicken embryos have been used as models to study cell migration, tissue patterns, tissue symmetry, vasculogenesis, and specific organ system biology, including cardiac morphogenesis, because of their advantages such as ease of in ovo visualization, ease of manipulation, low cost, well-characterized properties, and amenability to new molecular tools [30].Although chicks may not be as genetically tractable as mice for simulating syndromic CHD, they remain a useful model for studying structural cardiac diseases.However, it may not always be possible to accurately replicate abnormal cardiogenesis in chicks for human CHD patients because of the differences in certain cardiac events between chicks and humans, such as the development of the septum secundum and pharyngeal arch artery system [31].

Mice:
Cardiovascular disease (CVD) is best studied in mouse models, as it has a four-chambered heart and is evolutionarily more closely related to humans than flies or zebrafish [32].Studies in mice have shown that more than 500 mutated genes contribute to heart defects [33].Among these abnormalities, the genetic interaction between Tbx5 and Mef2c causes ventricular septation defects in transgenic mice [34].A comprehensive understanding of the genes, mutations, and underlying mechanisms responsible for the onset and progression of hereditary and de novo CHD in humans remains incomplete.An additional investigation using a zebrafish model confirmed the role of a rare causative gene in congenital cardiomyopathy, which leads to a fatal restrictive phenotype [47].This study used whole-exome sequencing and linkage analysis to investigate the genetic underpinnings of a newly characterized cardiac disorder in a Caucasian family.The family consisted of both unaffected and affected individuals, including a pair of twins.Researchers identified two genetic variations in KIF20A and conducted experiments using zebrafish embryos to investigate the effects of reducing KIF20A expression through MO-mediated knockdown.
The results showed that the zebrafish embryos with reduced KIF20A expression exhibited a progressive cardiac phenotype characterized by red blood cells near the atrium, increased heart rate, and cardiac edema suggesting that KIF20A plays an important role in heart development and function [47].Despite these advances, the use of zebrafish as a disease model has several limitations.The lack of cross-reactivity between mammalian and zebrafish antibodies limits the use of zebrafish as a model organism in protein biochemistry.Duplicated genes exhibit sub-functionalization, which complicates genetic analysis but allows for the study of several gene functions using mutants.The lack of embryonic stem cells for reverse genetic methods, such as knockout strain creation, has slowed scientific research on this organism [48].

Conclusion:
Advances in epidemiological research have led to a significant increase in the global prevalence of CHD, whereas genetic studies have shed light on various genetic abnormalities associated with different types of CHD.Therefore, understanding the genetics of CHD is crucial to improve its management and treatment.Studies on CHD genes have encompassed several models and methods.Animal models, both genetically engineered and naturally occurring, have played a significant role in elucidating the genetic basis of CHD.These models, including primates, canines, frogs, rabbits, chicks, mice, Drosophila, and zebrafish, have provided insights into the molecular mechanisms of cardiac development and effects of genetic mutations.Primates offer a high degree of genetic similarity to humans; however, their limited availability and high costs have limited their widespread use.Canine dogs have a cardiac structure comparable to that of humans; however, their cost is significant.Xenopus frogs are a practical model owing to their abundant embryos, affordability, and genetic manipulability.However, pseudotetraploidy in X. laevis and the functional redundancy among genes pose challenges.Rabbits have great potential as CHD models because of their similar cellular electrophysiology to humans; however, limitations in genetic manipulation and reproductive rates exist.Chickens offer valuable insights owing to their ease of manipulation and low cost, but differences in certain cardiac events compared to humans exist.Mice with four-chambered hearts and extensive genetic resources are a promising model.However, variations in physiology and genomics have also been reported.Fruit flies share genetic parallels with humans; however, differences in cardiac structure and open circulatory systems present hurdles.Zebrafish, with their genetic conservation, exhibit physiological similarities to the human heart, but face challenges such as a scarcity of cross-reactivity with mammalian antibodies and gene duplication.Recent advancements in induced iPSCs, hPSCs, and CRISPR/Cas9 have significantly affected this field.Each model has distinct advantages and disadvantages.iPSCs maintain the genetic of affected individuals, but are limited to 2D cell culture and genomic stability concerns.hPSCs can differentiate into cardiovascular cells, raising concerns regarding their genomic stability and imprinting loss.CRISPR-Cas9 technology is promising for correcting pathogenic mutations; however, offtarget effects remain an issue.The advantages and disadvantages of this method are summarized in Figure 2. The choice of method or model for CHD gene research is determined by the specific research goals, available resources, and ethical considerations.Researchers must carefully evaluate these advantages and disadvantages to select the most suitable approach for their studies.It is important to recognize that there is no ideal animal model for the human cardiovascular system and relying on only one animal model to address all issues is not advisable.Future research should embrace interdisciplinary approaches to untangle the complex genetic landscape of CHD, ultimately leading to the development of more effective diagnostic tools and therapeutic interventions.

Figure 1 :
Figure 1: In vitro and in vivo models to study the congenital heart diseases.

Figure 2 :
Figure 2: Advantages and disadvantages of congenital heart disease models.In vitro models: Induced pluripotent stem cells: Induced pluripotent stem cells (iPSCs) can be derived from adult somatic cells by forced reprogramming to differentiate into almost all cell types [49].Using patient-derived iPSCs offers a distinctive opportunity to investigate the genetic underpinnings of CHD as these cells maintain the complete genetic repertoire of the corresponding affected individuals.The integration of CRISPR/Cas9 genome editing, single-cell genomics, and cardiac organoid engineering techniques with iPSCs could serve as a valuable addition to existing mouse genetic models of CHD.Cardiomyocytes (CMs), vascular smooth muscle cells (SMCs), and endothelial/endocardial cells (ECs) derived from iPSCs can be used as human iPSC models of CHD [38].Wang et al. used CMs produced from iPSC-CMs obtained from individuals with Barth syndrome to characterize many metabolic, structural, and functional irregularities linked to TAZ mutations.The data presented in this study suggest that the overproduction of reactive oxygen species (ROS) plays a role in the development of sarcomere disarray and decreases contractile stress generation in Barth syndrome (BTHS) iPSC-CMs.The involvement of ROS in CM development, sarcomerogenesis and contractility is known [50].Patient-specific iPSC-CMs generated from patients with left ventricular non-compaction (LVNC) carrying a mutation in the cardiac transcription factor TBX20 are associated with perturbed transforming growth factor beta (TGF-β) signaling and a pathological LVNC phenotype at the single-cell level.In this study, TBX20 mutation was a probable causative agent of LVNC [51].In 2019, Gifford et al. used human iPSCs to learn about CHD, especially to validate MKL2, MYH7, and NKX2-5 genes.Data revealed that NKX2-5 variations have been identified as a genetic modifier in cases of LVNC cardiomyopathy, where the age at which symptoms manifest might range from childhood to the incidental discovery of asymptomatic cases in adults,

Table 1 :
Genes Associated with non-syndromic CHD

Table 4 [36].
Spielmann et al., 2022 screened 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities and identified approximately 705 lines with ventricular dilation, cardiac arrhythmia, and/or myocardial hypertrophy [35].The validated genes are listed in

Table 2 : Summarizes the mouse models of CHD
[39] in spindle assembly and cell cycle pathways of cardiomyocytes, which can affect cardiac development[37].Although animal models provide the most accurate representation of the in vivo environment, it is important to note that animals differ from humans in terms of their physiology and genomics.Therefore, these factors may not always be clinically relevant[38].The challenge of applying findings from animal studies to humans is due to the differences between species and variations across species.Therefore, the validity of preclinical animal studies is essential for extrapolation.External validity includes controllable factors, such as animal sample representativeness, relevance of animal models to therapy, and unchangeable features, such as differences between animal and human species[39].adulthood [42].Zhu et al. (2017) utilized a Drosophila melanogaster model and high-throughput in vivo functional validation of candidate CHD genes (Table 5) [43].

Table 5 : Validated CHD-associated genes and their Drosophila analogs
[46]afish are particularly sensitive to small-molecule treatments and are thus suitable for chemical genetic studies and screening to identify additional cellular and molecular pathways that may regulate cardiovascular development.Through precise genome editing using single-stranded oligodeoxynucleotides, researchers have introduced the human PBX3 p.A136V variant into zebrafish pbx4 using CRISPR-Cas9 genome editing[46].This study was performed to investigate whether this variant, which is more common in patients with CHD, acts as a genetic modifier in zebrafish heart development.The results showed that the pbx4 p.A131V variant could enhance myocardial morphogenesis defects caused by loss of hand2, a cardiac specification factor.These findings suggest that the pbx4 p.A131V allele may be a genetic modifier of the heart[46].
5 variations have been identified as a genetic modifier in cases of LVNC cardiomyopathy, where the age at which symptoms manifest might range from childhood to the incidental discovery of asymptomatic cases in adults, These include the possibility of off-target effects, restricted genome-targeting scope due to protospacer-adjacent motif sequences, and suboptimal efficiency and specificity.Consequently, numerous research teams have endeavored to enhance this technology [65].
[64]o and in vivo[63].The main CHD-causing genes that were discovered or validated using CRISPR/Cas9 are listed in Table 6[64].Regrettably, certain constraints persist in CRISPR-Cas systems, which require resolution.